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John Clancy, Jr., Ph.D.
Host Defense – 2012
Tissues and Organs of the Immune System
TISSUE AND ORGANS OF THE IMMUNE SYSTEM
Date: March 9, 2012 - 9:30 AM & March 13, 2012 - 10:30 AM
LEARNING GOALS
You will be able to describe the location, structure and general function of the thymus, lymph nodes,
spleen and mucosal associated lymphoid tissue.
OBJECTIVES
To attain the goal of this lecture you will be able to:
• State the difference between central (primary) versus peripheral lymphoid organs.
• Describe the development, structure and general function of the thymus.
• Draw the general sequence of events in CD4 and CD8 lymphocyte development from thymocytes.
Understand where positive and negative selection occurs and its consequences.
Describe where double negative, double positive, and single positive cells are found.
• Describe the structure and function of lymph nodes.
• Diagram the path taken by lymph versus blood as they flow through lymph nodes. Understand the
similarities and differences between T and B cell circulation.
• Describe the structure and function of the spleen.
• Compare and contrast the path taken by T and B cells as they circulate through lymph nodes and
spleen.
• Describe the different classes of lymphoid tissue associated with the gastrointestinal tract (GALT).
READING ASSIGNMENTS
Junqueira, et al, Basic Histology, 11th Edition, Chapter 14.
Wheater’s Functional Histology, 5th Edition, Chapter 11.
Janeway, et al, Immunobiology, 7th Edition, Chapters 1 (pp.3-13), 7 (pp.273-280), 8 (pp. 325327), 9 (pp. 387-390).
LECTURER
John Clancy, Jr., Ph.D.
1
John Clancy Jr., Ph.D.
Host Defense - 2012
Tissues and Organs of the Immune System
I.
CENTRAL VERSUS PERIPHERAL LYMPHOID ORGANS (Fig. 1.7)
•
1° or Central: Bone marrow, Thymus
•
2° or Peripheral: Lymph nodes, Spleen, Tonsils, Peyers Patches, Appendix
•
3°: Where immune responses occur outside lymphoid organs. E.g. Pannus in an arthritic
joint.
•
Stroma: Reticular cells and fibers (Type III Collagen) except thymus
•
Parenchyma: Lymphocytes, macrophages, dendritic cells, NK cells (spleen)
Fig. 1.7 The distribution of lymphoid
tissues in the body. Lymphocytes arise from
stem cells in bone marrow, and differentiate
in the central lymphoid organs, B cells in
bone marrow and T cells in the thymus.
Author:
Janeway,
al tissues and are
They migrate
frometthese
From:
Immunobiology,
6thtoedition
carried
in the bloodstream
the peripheral or
Reproduced
by permission
Routledge,
secondary lymphoid
organs of
, the
lymph
Inc.
nodes, spleen, and lymphoid tissues
associated
with mucosa,
likeGroup
the gutPart
of The Taylor
Francis
associated tonsils, Peyer’s patches, and
appendix. The peripheral lymphoid organs
are the sites of lymphocyte activation by
antigen, and lymphocytes recirculate between
the blood and these organs until they
encounter their specific antigen. T cells
receive survival signals from dendritic cells
(DCs) in the periphery, whereas the source of
the survival signals for B cells is thought to
be in the lymphoid follicles. Lymphatics
drain extracellular fluid from the peripheral
tissues, through the lymph nodes and into the
thoracic duct, which empties into the left
subclavian vein. This fluid, known as lymph,
carries antigen taken up by DCs and
macrophages to the lymph nodes and
recirculating lymphocytes from the lymph
nodes back into the blood. Lymphoid tissue
is also associated with other mucosa such as
the bronchial linings (not shown).
Author: Janeway, et al
From: Immunology 7th Edition
Reproduced by permission of Routledge, Inc.
Part of The Taylor Francis Group
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John Clancy Jr., Ph.D.
Host Defense - 2012
Tissues and Organs of the Immune System
II.
THYMUS: ENCAPSULATED WITH EPITHELIAL STROMA
(Figs. 7.15 and 7.21)
•
Origin: Ventral part of 3rd pharyngeal pouch and third branchial cleft. Endodern and
underlying mesoderm origin. Two lobes.
•
Capsule of Fibroblasts that form trabeculae or septa which separate the cortex into lobules.
Each lobule connected to a central running confluent medulla.
•
Stroma of Epithelial Reticular Cells which secrete IL-7 and contain tonofilaments.
¾ Cortex:
Developing T cells: CD3-4-8- → γ:δ+ (T cell receptor) CD3+ or α:β (T cell
receptor) CD3+4+8+. CD3 is present on all T cells and acts in signal
transduction.
-
Nurse cells (subcapsular epithelial cells); surround CD3- 4-8- cells and
secrete IL-7.
-
Cortical Epithelial Cells which make contact with CD3+4+8+ cells and
display self antigens.
¾ Cortico-Medullary Junction
Dendritic cells (Bone Marrow Derived): make contact with CD3+4+8+.
-
Macrophages make contact with CD3+4+8+; ingest apoptotic cells
¾ Medulla:
Hassall's Corpuscles (Whorl-shaped Epithelial Cells): no known function
but only found in the thymus. Increase with age.
•
Mature T cells: CD3+4+ or CD3+8+
Parenchyma: Thymocytes
- Origin: pro-T from marrow: CD3-4-8- which localize in the subcapsular cortex,
where they are surrounded by IL-7 secreting nurse cells.
-
Cortex contains 80-90% of all thymocytes.
ƒ
Superficial (subcapsular): 5% of all thymocytes which are mainly large
blasts: CD3-4-8- cells proliferate for 1 week.
ƒ
Mid-Cortex to Cortico-Medularry junction: CD3+4+8+ mature for 2
weeks.
+ and then - selection eliminate unnecessary or potentially
autoreactive cells via apoptosis as no inflammation. This is a
selection process where thymocytes must recognize self MHC antigens
but not too well.
-
-
Medulla: 10% of all thymocytes: CD3+4+ or CD3+8+ mature cells which exit to
peripheral lymphoid organs.
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John Clancy Jr., Ph.D.
Host Defense - 2012
Tissues and Organs of the Immune System
Fig. 7.15 The cellular organization of the human thymus. The thymus, which lies in the midline of the body, above the heart, is made
up of several lobules, each of which contains discrete cortical (outer) and medullary (central) regions. As shown in the diagram, the
cortex consists of immature thymocytes, branched cortical epithelial cells, with which the immature cortical thymocytes are closely
associated, and scattered macrophages, which are involved in clearing apoptotic thymocytes. The medulla consists of mature thymocytes,
and medullary epithelial cells, along with macrophages and dendritic cells of bone marrow origin. Hassall’s corpuscles are probably also
sites of cell destruction. The thymocytes in the outer cortical cell layer are proliferating immature T cells undergoing thymic selection.
The photograph shows the equivalent section of a human thymus, stained with hematoxylin and eosin. The cortex is darkly staining; the
medulla is lightly stained. The large body in the medulla is a Hassall’s corpuscle. Photograph courtesy of C.J. Howe.
Fig. 7.21 Thymocytes at different
developmental stages are found in
distinct parts of the thymus. The
earliest cells to enter the thymus are
found in the subcapsular region of the
cortex. As these cells proliferate and
mature into double-positive thymocytes,
they migrate deeper into the thymus
cortex. Finally, the medulla contains
only mature single-positive T cells,
which eventually leave the thymus and
enter the bloodstream.
Copyright: 2008
Author: Janeway, et al
From: Immunology, 7th edition
Reproduced by permission of
Routledge, Inc., part of the
Taylor Francis Group
4
III.
John Clancy Jr., Ph.D.
Host Defense - 2012
Tissues and Organs of the Immune System
•
Proliferation
¾ >any other lymphoid organ but under endogenous control as not antigen driven.
¾ <4% of all thymocytes produced after three weeks leave for periphery as CD3+4+ or
CD3+8+ lymphocytes. Most cells die via apoptosis. Twice as many CD3+4+ survive than
CD3+8+.
•
Thymosin or Thymopoietin hormone produced by thymic epithelial cells. This concept
largely replaced by epithelial derived cytokines → IL-7, IL-2 and IL-6 necessary for
thymocyte development. Adrenocortico steroids decrease cortical T cell numbers.
•
Young vs. Old: The thymus is well developed at birth but looses much of its parenchyma as
we age. A decrease in cellularity is particularly evident after puberty. Nevertheless, recent
evidence indicates that even older thymuses are functional.
•
Weakly permeable protective environment in the thymus such that most circulating antigens
are excluded from developing immature cortical cells. Blood-thymus barrier.
•
NK (CD56) cells develop in the bone marrow and diverge early from the pro-T/NK pathway
and are not thymus dependent.
LYMPH NODE: ENCAPSULATED WITH TYPE I COLLAGEN WITH RETICULAR
CELL AND FIBER (Type III Collagen) STROMA ATTACHED. (Figs. 14.20, 1.18, 4.17,
1.9, 9.9, 9.10)
•
Trabecula extend from capsule and separate cortex into superficial outer cortex with
follicles and an inner paracortex or deep cortex. An inner medulla leads to a hilus where
blood vessels and the efferent lymphatic is found.
•
Parenchyma made up of lymphocytes in the superficial follicles and homogeneous cortex
which leads to medullary cords lined by medullary lymph sinuses.
•
Reticular stroma creates lymph sinuses which are called subcapsular, trabecular and
medullary.
•
Lymph circulation (Fig 14-20).
¾ Afferent lymphatics receive lymphatic vessels from the periphery. Have valves directed
to the subscapular sinus.
¾ Subcapsular → Trabecular → Medullary Sinuses → Efferent Lymphatic →
Thoracic Duct. Sinues made up of stellate reticular cells and fibers. Macrophages
extensively attach.
Which contains:
- Lymphocytes (B & T)
- Lymphoblasts (B & T)
- Macrophages
•
Blood Circulation
¾ Hilar artery → Cortical nodule capillaries → Deep Cortical Post - Capillary venules with
a high endothelium (HEPCV) → Hilar vein
-
It is through the HEPCVs that the main players of the Adaptive Immune System
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John Clancy Jr., Ph.D.
Host Defense - 2012
Tissues and Organs of the Immune System
(T and B cells) gain entrance into the lymph node from the circulation.
•
Cortex
¾ Superficial
-
1° nodule (follicle): B cells mainly
-
2° nodule with germinal center are the sites where B cells mature with the help of
CD4+ T cells into either Plasmablasts or Memory Cells (Fig. 1.18, 9.10)
ƒ
Macrophages
ƒ
Follicular Dendritic Cell which retain antigen for long periods
ƒ
Small and Large Lymphocytes (1°B, some T)
ƒ
Peripheral corona (mantle) surrounds 2o nodule and is a migration zone.
Basic Histology 11th Edition
Author : Junqueira, et. Al
Reproduced with permission of the
McGraw-Hill Companies
Figure 14-20. Schematic representation of the blood and lymph circulation in a lymph node. Note that
lymph enters through the convex side of the node and leaves through the hilum. Blood enters and leaves
the node by the hilum.
Fig. 1.18 Organization of a lymph node. As shown in the diagram on the left, a lymph node consists of an outermost cortex and an inner medulla. The
cortex is composed of an outer cortex of B cells organized into lymphoid follicles and deep, or paracortical, areas made up mainly of T cells and dendritic
cells. When an immune response is underway, some of the follicles contain central
6 areas of intense B-cell proliferation called germinal centers and are known
as secondary lymphoid follicles. These reactions are very dramatic, but eventually die out as senescent germinal centers. Lymph draining from the
extracellular spaces of the body carriers antigens in phagocytic dendritic cells and phagocytic macrophages from the tissues to the lymph node via the afferent
lymphatics. Lymph leaves by the efferent lymphatics in the medulla. The medulla consists of strings of macrophages and antibody-secreting plasma cells
known as the medullary cords. Naïve lymphocytes enter the node from the bloodstream through specialized postcapillary venules (not shown) and leave with
John Clancy Jr., Ph.D.
Host Defense - 2012
Tissues and Organs of the Immune System
Fig. 9-10 Germinal centers are formed when activated B cells enter lymphoid follicles. The germinal center is a specialized microenvironment in which Bcell proliferation, somatic hypermutation, and selection for antigen binding all occur. Closely packed centroblasts from the so-called “dark zone” of the
germinal center, as can be seen in the lower part of the photomicrograph in the center, which shows a high-power view of a section through a human tonsillar
germinal center. The photomicrograph on the right shows a lower-power view of a tonsillar germinal center; B cells are found in the dark zone, light zone, and
mantle zone. Proliferating cells are stained green for Ki67, an antigen expressed in nuclei of dividing cells, revealing the centroblasts in the dark zone. The
dense network of follicular dendritic cells, stained red, mainly occupies the light zone. Cells in the light zone are also proliferating, though to a lesser degree in
most germinal centers. Small recirculating B cells occupy the mantle zone at the edge of the B-cell follicle. Large masses of CD4 T cells, stained blue, can be
seen in the T-cell zones, which separate the follicles. There are also significant numbers of T cells in the light zone of the germinal center; CD4 staining in the
dark zone is mainly associated with CD4-positive phagocytes. Photographs courtesy of I. MacLennan.
FOLLICULAR MANTLE
Storage of long-lived
Memory B Cells
APICAL LIGHT ZONE
Differentiation of selected
Centrocytes
BASAL LIGHT ZONE
Apoptotic death
DARK ZONE
Proliferation of
centroblasts
John Clancy, Jr., PhD
- Germinal Center Zonation: activated B cells called Centroblasts or
Centrocytes (Fig. 9.10)
ƒ
Dark Zone at the base: Centroblasts (B blasts) without surface
immunoglobulin (SIG). Hypermutation occurs to make more avid
antibody.
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John Clancy Jr., Ph.D.
Host Defense - 2012
Tissues and Organs of the Immune System
-
•
ƒ
Basal Light Zone: Centrocytes (B cells) with SIG. Antigen-bearing
follicular dendritic cells (FDCs). Centrocytes that bind antigen on
FDCs survive, those that don’t die by apoptosis.
ƒ
Apical Light Zone: Differentiation of surviving centrocytes to
become B Blast (Plasmablast) or Memory Cells
ƒ
Migration and Differentiation of some B blasts into the Medullary
Cords to become Plasma Cells (short lived). Long-lived
plasmablasts leave via the efferent lymphatics and go to the bone
marrow. (Figure 4.17).
ƒ
Follicular Mantle: Memory Cells and some non-reactive B cells.
Inner or paracortex
ƒ
Diffuse cells: 1° T, some B
ƒ
Langerhans Cell (Immature Dendritic Cell) originates in the marrow
and localizes in the epidermis of the skin. It brings processed antigen
into the node from periphery via afferent lymphatics and becomes a
mature antigen presenting cell called an Interdigitating Dendritic Cell.
It presents antigen to T cells. (Fig 1.19)
ƒ
High endothelial post-capillary venule (HEPCVs) function as the gate
of entry of T and B cells from the circulation into the lymph node.
Medulla (Figs 4.17, 9.9).
¾ Cords
- Contains Plasmablasts and plasma cells which live10-20 days; long lived
Plasmablasts enter the efferent lymph and go to bone marrow. Plasmablasts
that will differentiate into IgA producing plasma cells go to the lamina
propria of the gut.
¾ Medullary Sinuses drain into the efferent lymphatics.
8
John Clancy Jr., Ph.D.
Host Defense - 2012
Tissues and Organs of the Immune System
Modified from:
The Immune System
2nd edition
Author: P. Parham
Publisher: Garland
Reproduced by permission
of the Garland Companies
Figure 4.17 B cells encountering antigens in
secondary lymphoid tissues form germinal
centers and undergo differentiation to plasma
cells. A lymph node is illustrated here. A B cell
entering the lymph node through a HEV
encounters antigen in the lymph node cortex.
Antigen was delivered in the afferent lymph that
Drained from infected tissue. The B cell is
drained from infected tissue. The B cell is
activated by CD4 helper T cells in the T-cell areas
to form a primary focus of dividing cells. From
this, some B cells migrate directly to the medullary
cords secreting plasma cells. Other B cells migrate
into a primary follicle to form a germinal center. B
cells continue to divide and differentiate within
the germinal center. Activated B cells migrate
from the germinal center to the medulla of the
lymph node or to the bone marrow to complete
their differentiation into plasma cells.
Fig. 1.9 Dendritic cells initiate
adaptive immune responses.
Immature dendritic cells resident in
a tissue take up pathogens and their
antigens by macrophinocytosis and
by receptor-mediated endocytosis.
They are stimulated by recognition
of the presence of pathogens to
migrate through the lymphatics to
regional lymph nodes, where they
arrive as fully mature
nonphagocytic dendritic cells that
express both antigen and the
necessary surface molecules to
stimulate clonal expansion. In the
lymph node, the mature dendritic
cell encounters and activates
antigen-specific naïve T
lymphocytes, which enter the node
from the blood through a
specialized blood vessel known
from its cubodial endothelial cells
as a high endothelial venule (HEV).
Janeway 6th Edition
9
John Clancy Jr., Ph.D.
Host Defense - 2012
Tissues and Organs of the Immune System
Fig. 9.9 Activated B cells form germinal centers in lymphoid
follicles. Some B cells activated at the T-cell/B-cell border migrate to
form a germinal center within a primary follicle. Germinal centers
are sites of rapid B-cell proliferation and differentiation. Follicles in
which germinal centers have formed are known as secondary follicles.
Within the germinal center, B cells begin their differentiation into either
Antibody-secreting plasma cells or memory B cells. Plasma cells leave
The germinal center and migrate to the medullary cords or leave the lymph
Node altogether via the efferent lymphatics and migrate to the bone marrow.
Memory B cells continue to recirculate through the B-cell zones of secondary
lymphoid tissue (not shown) and some may preferentially reside in the splenic
marginal zone as described in Chapter 7.
Copyright: 2008
Author: Janeway, et al
From: Immunobiology, 7th edition.
Reproduced by permission of Routledge, Inc.
Part of The Taylor Francis Group.
10
John Clancy Jr., Ph.D.
Host Defense - 2012
Tissues and Organs of the Immune System
IV.
SPLEEN: ENCAPSULATED WITH RETICULAR STROMA
•
Connective tissue trabecula extend from capsule and ensheath splenic blood vessels forming
Trabecular Arteries and Veins. Reticular fiber network attached to capsule and trabeculae
forming a framework or stroma.
•
White pulp (Fig.14-28) is lymphoid tissue
•
¾
Splenic nodules 1° and 2° nodules like lymph node
¾
Central artery comes from trabecular artery
¾
Periarteriolar lymphoid sheath (PALS): 1°T cells ensheathing central artery
Marginal zone contains memory B cells, T independent B cells (polysaccharides of
bacterial cell walls), and macrophages. Separates white and red pulp.
¾
•
Marginal zone blood sinuses are found at the periphery of the nodules and receive
T and B cells from small branches of the central artery.
Red pulp (RP)
¾
Cords of Bilroth (RP cords) which can extend from the white pulp:
- Contain Hematopoietic tissue and Plasma Cells and are separated by blood sinusoids
•
¾
Sinusoids are lined by endothelial cells with holes (2-3 um wide) in them and an
incomplete (discontinuous) basal lamina as in the bone marrow. Many macrophages
attached.
¾
Penicillar arteries come from central arteries and may empty into sinusoids or RP
cords.
Open versus closed (continuous) circulation refers to whether penicillar arteries:
1) Open → parenchyma of RP cords and then into sinusoids, or
2) Closed → directly into sinusoids
y
Old RBCs lose surface sialic acid exposing surface galactose which are phagocytosed by
sinusoid adhering macrophages.
y
Hemopoietic in second trimester and some disease states.
11
John Clancy Jr., Ph.D.
Host Defense - 2012
Tissues and Organs of the Immune System
Basic Histology 11th Edition
Author: Junqueira, et. Al
Reproduced with permission of
The McGraw-Hill Companies
Figure14-28 Schematic view of the blood circulation of the spleen. Theories of open and close circulation are
Represented. Splenic sinuses (S) are indicated. PALS, periarterial lymphatic sheath.
V.
GASTROINTESTINAL TRACT
•
•
Epithelial lining contains CD3+8+αβ and CD3+ γδ cells to moniter luminal antigens.
Diffuse, in lamina propria just underneath the epithelia lining throughout the GI tract.
-
1° nodule: Ig A producing B cells
-
2° nodule with germinal center
1° B cells, some T cells as well as same cells found in lymph node and spleen
germinal centers
ƒ
•
T Cells (αβ, CD3+)
Partially encapsulated and covered by epithelium. Present in lamina propria and submucosa
of certain areas of GI tract.
¾
Peyer's Patches (Ileum) (Fig. 1.20, 10.19, and 14-40)
-
Villi and Domes which overlie lymphoid tissue
-
Domes are covered by M cells: Epithelial cells which become antigen
transporting. They are derived from surface lining epithelium and require B cells
for their development. Also found in the lung.
12
John Clancy Jr., Ph.D.
Host Defense - 2012
Tissues and Organs of the Immune System
Figure 1.20. A typical region of gut-associated lymphoid tissue. A schematic diagram (left panel) and a light
micrograph (right panel) and a light micrograph (right panel) of a typical region of gut-associated lymphoid tissue. M
cells of the gut epithelial wall deliver pathogens from the luminal side of the gut mucosa to the lymphoid tissue within
the gut wall. These areas are organized similarly to the lymph node and the white pulp of the spleen, with distinctive Band T-cell zones, lymphoid follicles, and germinal centers. Photograph courtesy of N. Rooney. Janeway 6th Edition
Fig. 10.19 M cells take up antigens from the lumen of the gut by endocytosis. The cell
membrane at the base of these cells is folded around lymphocytes and dendritic cells within the
Peyer’s patches. Antigens are transported through M cells by the process of transcytosis and
delivered directly to antigen-presenting cells and lymphocytes of the mucosal immune system.
13
John Clancy Jr., Ph.D.
Host Defense - 2012
Tissues and Organs of the Immune System
-
Intraepithelial lymphocytes (CD3+8+ αβ and CD3+γδ) found here as well as all
along the GI tract.
-
Lymphoid nodules (10 and 20) with B and T cells as in other lymphoid organs.
-
Internodular (Interfollicular) tissue contain high endothelial venules and 1° T cells
as in the paracortex of a lymph node and PALS of the spleen. T and B cells enter
the Peyer’s Patches here as HEVs found here.
Basic Histology 11th
Edition
Author:Junqueira, et. Al
Reproduced with
permission
McGraw-Hill
Companies
Figure 14-11, p. 265 General view of the mucosa immunity in the intestine. Luminal antigens are
captured by dome-shaped M cells present in the covering of Peyer’s patches and transported to subjacent
lymphocytes, macrophages, and dendritic cells. Macrophages and dendritic cells migrate to neighboring
lymph nodes, where they stimulate B and T lymphocytes, which then enter the lymphatic circulation and
later the blood circulation (lymph flows to the blood). The stimulated lymphocytes home in other tissues,
including the mucosa lamina propria, where plasma cells produce considerable amounts of IgA. The
lymphoid cells of the lamina propria of intestional mucosa are a major anina propria of intestional mucosa
are a major antibody producer, because of their extension and close contract with antigens introduced into
the digestive tract.
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John Clancy Jr., Ph.D.
Host Defense - 2012
Tissues and Organs of the Immune System
¾ Appendix at the beginning of the large intestine.
- No villi but lamina propria and submucosal lymphoid tissue.
•
Tonsils (Fig. 14-26)
¾ Palatine: lined by stratified squamous nonkeratinizing epithelium. Between
palatoglossal and palatopharyngeal folds.
- Many (10-12) deep crypts (Fig. 14-26)
- Capsule underneath
- Skeletal muscle deep to capsule
¾ Pharyngeal (Adenoids): pseudostratified ciliated columnar with some stratified
squamous; no crypts but shallow pleats. Roof of nasal pharynx.
¾ Lingual: stratified squamous, single crypt.
•
Mucosal Associated Lymphoid Tissue (MALT) is found under all epithelial linings of
mucosa (respiratory, gut, urinary, etc.)
•
Gut Associated Lymphoid tissue ((GALT) is thus only a part of the MALT system
Basic Histology 9th Edition
Junqueira, et Al
with permission of the
Companies
Author:
Reproduced
McGraw-Hill
Figure 14-26. Palatine tonsil. Numerous lymphoid nodules can be seen near the stratified squamous
epithelium of the oropharynx. The light areas in the lymphoid tissue are germinal centers. Note the sections
Through the epithelial crypt (C).
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John Clancy Jr., Ph.D.
Host Defense - 2012
Tissues and Organs of the Immune System
VI.
LYMPHOCYTE TRAFFIC (Fig. 1.16)
•
Lymphocytes circulate from the blood into various tissues and lymphoid organs and back to
the blood continuously. 5 X 1011 leave the circulation to enter the spleen and return into the
blood per day.
•
Lymphocytes leave the blood thru HEV in lymph nodes and Peyer’s Patches. They
eventually enter efferent lymphatics and the thoracic duct system which joins the left
subclavian and internal jugular veins. 0.3 X 1011 return to the blood from the lymph per day.
•
Lymphocytes enter the white pulp of the spleen from the marginal zone blood sinuses. They
eventually find their way into the red pulp blood sinuses and leave via the splenic vein.
Patterns of lymphocyte traffic
Janeway 7th Edition
Figure1.17 Circulating lymphocytes meet lymph-borne pathogens in draining lymph nodes. Lymphocytes leave the blood
and enter lymph nodes, where they can be activated by pathogens draining in the afferent lymph from a site of infection. The
circulation pertaining to a site of infection in the left foot is shown. When activated by pathogens, lymphocytes stay in the node
to divide and differentiate into effector cells. If lymphocytes are not activated, they leave the node in the efferent lymph and are
carried by the lymphatics to the thoracic duct, which empties into the blood at the left subclavian vein. Lymphocytes recirculate
all the time, irrespective of infection. Every minute 5x106 lymphocytes leave the blood and enter secondary lymphoid tissues.
16
Tissue and Organs
of
The Immune System
John Clancy, Jr., Ph.D.
Janeway, 7th Edition
I. Central Versus Peripheral Lymphoid
Organs (Fig. 1.7)
• 1° or Central: Bone Marrow, Thymus
• 2° or Peripheral: Lymph nodes, Spleen,
Tonsils, Peyer’s Patches, Appendix
• 3° Where immune responses occur
outside lymphoid organs, E.g. Pannus
in an arthritic joint.
1
• Stroma: Reticular cells and fibers (Type III
Collagen) except thymus.
• Parenchyma: Lymphocytes, macrophages,
dendritic cells, NK cells (spleen)
Lymph Node
Silver Stain:  Type III Collagen
John Clancy, Jr. PhD
Young and Old Thymus
John Clancy, Jr., PhD
2
II. Thymus: Encapsulated with Epithelial
stroma (Figs. 7.15 and 7.21)
• Origin: Ventral part of 3rd pharyngeal
pouch and third branchial cleft.
Endoderm and underlying mesoderm
origin. Two lobes.
• Capsule of Fibroblasts that form
trabeculae or septa which separate the
cortex into lobules. Each lobule
connected to a central running confluent
medulla.
Young Thymus
Cortex
Medulla
John Clancy, Jr., PhD
Junqueira 11th Edition, Fig 14-16
3
• Stroma of Epithelial Reticular Cells
which secrete IL-7 and contain
tonofilaments .
Nurse Cells
Janeway 7th Edition
Cortex
Medulla
John Clancy, Jr., PhD
4
Hassall’s Corpuscle
John Clancy, Jr., PhD
• Parenchyma: Thymocytes
– Origin: pro-T from marrow: CD3-,4-,8which localize in the subcapsular cortex,
where they are surrounded by IL-7 secreting
nurse cells
• cortex contains 80-90% of all thymocytes.
– Superficial (subcapsular): 5% of all
thymocytes which are mainly large blasts:
CD3-,4-,8- cells proliferate for 1 week
5
Mt: Mitosis
C: Capsule
Wheater’s 5th Ed. Fig 11.6
• Mid-Cortex to Cortico-Medullary junction:
CD3+4+8+ mature for 2 weeks.
- + and then – selection eliminate
unnecessary or potentially autoreactive cells
via apoptosis as no inflammation. This is a
selection process where thymocytes must
recognize self MHC antigens but not too well.
CD3,4,8
CD3,4
or
CD3,8
John Clancy, Jr. PhD
6
- Medulla: 10% of all thymocytes: CD3+4+ or
CD3+8+ mature cells which exit to
peripheral lymphoid organs.
Janeway 6th Edition
7
• Proliferation
 >any other lymphoid organ but under
endogenous control as not antigen
driven.
 <4% of all thymocytes produced after
three weeks leave for periphery as
CD3+4+ or CD3+8+ lymphocytes. Most
cells die via apoptosis. Twice as many
CD3+4+ survive than CD3+8+.
• Young vs. Old: The thymus is well
developed at birth but looses much of its
parenchyma as we age. A decrease in
cellularity is particularly evident after
puberty. Nevertheless, recent evidence
indicates that even older thymuses are
functional.
Young Thymus
John Clancy Jr., PhD
8
Old Thymus
John Clancy Jr., PhD
Old Thymus
John Clancy Jr., PhD
• Weakly permeable protective
environment in the thymus such that
most circulating antigens are
excluded from developing immature
cortical cells. Blood-thymus barrier.
9
John Clancy Jr., PhD
• NK (CD56) cells develop in the
bone marrow and diverge early
from the pro-T/NK pathway and are
not thymus dependent.
,15
10
John Clancy Jr., PhD
III. Lymph node: Encapsulated with
Type I collagen with reticular cell
and fiber (Type III Collagen)
stroma attached.
Junqueira 11th Edition, Fig. 14-20
11
• Trabecula extended from capsule
and separate cortex into superficial
outer cortex with follicles and an
inner paracortex or deep cortex.
An inner medulla leads to a hilus
where blood vessels and the
efferent lymphatic is found.
c = Capsule
pc = paracortex
t = trabecula
co = medullary cord
ss = subcapsular sinus
John Clancy Jr., PhD
• Parenchyma made up of
lymphocytes in the superficial outer
cortex with follicles and an inner
paracortex or deep cortex. An inner
medulla leads to a hilus where blood
vessels and the efferent lymphatic is
found.
12
Reticular Stroma
John Clancy Jr., PhD
• Reticular stroma creates lymph
sinuses which are called
subcapsular, trabecular and
medullary.
Junqueira 11th Edition, Fig. 14-20
13
Afferent Lymphatic
John Clancy Jr., PhD
• Lymph circulation (Fig. 14-20).
 Afferent lymphatics receive
lymphatic vessels from the
periphery. Have valves directed
to the subcapsular sinus.
 Subcapsular → Trabecular → Medullary
Sinuses → Efferent Lymphatic →
Thoracic Duct. Sinus made up of stellate
reticular cells and fibers. Macrophages
extensively attach.
Which contains:
- Lymphocytes (B & T)
- Lymphoblasts (B & T)
- Macrophages
14
Junqueira 11th Edition, Fig. 14-20
• Blood Circulation
 Hilar artery → Cortical nodule
capillaries → Deep Cortical Post –
Capillary venules with a high
endothelium (HEPCV) → Hilar vein.
Superficial
Cortex
Deep
Cortex
with
HEPCV
John Clancy Jr., PhD
15
John Clancy, Jr., PhD
- It is through the HEPCVs that the
main players of the Adaptive
Immune System (T and B cells)
gain entrance into the lymph node
from the circulation.
HEPCV
Junqueira 11th Edition Fig. 14-26
16
• Cortex
 Superficial
- 1° nodule (follicle): B cells
mainly
1 Nodule
2 Nodule
John Clancy Jr., PhD
2 Nodule with Germinal Center within Arrowheads
John Clancy Jr., PhD
17
- 2° nodule with germinal center are the sites where
B cells mature with the help of CD4+ T cells into
either Plasmablasts or Memory Cells (Fig. 1.8, 9.12)
• Macrophages
• Follicular Dendritic Cell which retain antigen
for long periods
• Small and Large Lymphocytes (1°B, some T)
• Peripheral corona (mantle) surrounds 2°
nodule and is a migration zone.
Janeway 6th Edition
• Migration and Differentiation of some B
blasts into the Medullary Cords to
become Plasma Cells (short lived).
Long-lived plasmablasts leave via the
efferent lymphatics and go to the bone
marrow.
• Follicular Mantle: Memory Cells and
some non-reactive B cells.
18
Plasma blast
John Clancy Jr., PhD
- Inner or paracortex or deep cortex
• Diffuse cells: 1° T, some B
• Langerhans Cell (Immature Dendritic Cell)
originates in the marrow and localize in the
epidermis of the skin. It brings processed
antigen into the node from periphery via
afferent lymphatics and becomes a mature
antigen presenting cell called an
Interdigitating Dendritic Cell. It presents
antigen to T cell (Fig. 1.9)
• High endothelial post-capillary venule
(HEPCVs) function as the gate of entry of T
and B cells from the circulation into the lymph
node.
Janeway 6th Edition
19
• Medulla (Figs. 4.17, 9.9)
 Cords
- Contains Plasmablasts and plasma
cells which live 10-20 days; long
lived Plasmablasts enter the efferent
lymph and go to bone marrow.
Plasmablasts that will differentiate into
IgA producing plasma cells go to the
lamina propria of the gut.
 Medullary Sinuses drain into the efferent
lymphatics.
Janeway 7th Edition
Medulla
John Clancy Jr., PhD
20
Medullary Cords
Sinuses
John Clancy Jr., PhD
IV.
Spleen: Encapsulated with Reticular
Stroma
• Connective tissue trabecula extend from
capsule and ensheath splenic blood
vessels forming Trabecular Arteries and
Veins. Reticular fiber network attached
to capsule and trabeculae forming a
framework or stroma.
Junqueira 11th Edition Fig. 14-27
21
Junqueira 11th Edition Fig. 14-28
• White pulp (Fig. 14-28) is lymphoid tissue
 Splenic nodules 1° and 2° nodules like
lymph node.
 Central artery comes from trabecular
artery.
 Periarteriolar lymphoid sheath (PALS):
1°T cells ensheathing central artery.
Marginal Zone
White Pulp
Red Pulp
John Clancy Jr., PhD
22
• Marginal zone contains memory B cells, T
independent B cells (polysaccharides of
bacterial cell walls), and macrophages.
Separates white and red pulp.
 Marginal zone blood sinuses are found
at the periphery of the nodules and
receive T and B cells from small
branches of the central artery.
Silver Stain
John Clancy Jr., PhD
Junqueira 11th Edition, Fig. 14-28
23
• Red pulp (RP)
 Cords of Bilroth (RP cords) which
can extend from the white pulp:
- Contain Hematopoietic tissue and
Plasma Cells and are separated by
blood sinusoids
Red Pulp Cord
Red Pulp Vascular Sinus
John Clancy Jr., PhD
 Sinusoids are lined by endothelial cells
with holes (2-3 um wide) in them and an
incomplete (discontinuous) basal lamina
as in the bone marrow. Many
macrophages attached.
24
Junqueira Fig. 14-34
Junqueira Fig. 14-33
 Penicillar arteries come from
central arteries and may empty into
sinusoids or RP cords.
25
Junqueira Fig. 14-28
• Open versus closed (continuous)
circulation refers to whether penicillar
arteries:
1) Open → parenchyma of RP cords and
then into sinusoids, or
2) Closed → directly into sinusoids
• Old RBCs lose surface sialic acid
exposing surface galactose which are
phagocytosed by sinusoid adhering
macrophages.
• Hemopoietic in second trimester and some
disease states.
26
Red Pulp Macrophages
Junqueira Fig. 14-35
V. Gastrointestinal Tract
• Epithelial lining contains
CD3+8+ and CD3+ cells to
monitor luminal antigens.
Simple Columnar epithelia with intraepithelial lymphocytes
Diffuse lymphoid tissue in loose areolar CT of lamina propria
John Clancy Jr., PhD
27
• Diffuse, in lamina propria just
underneath the epithelia lining
throughout the GI tract.
Diffuse
lymphoid tissue
in LP of villi
John Clancy Jr., PhD
Isolated lymphoid
nodule
John Clancy Jr., PhD
28
- 1° nodule: IgA producing B cells.
- 2° nodule with germinal center
• 1° B cells, some T cells as well as
same cells found in lymph node and
spleen germinal centers
- T Cells (, CD3+)
P = Peyer’s Patch (PP)
d= Dome
John Clancy Jr., PhD
Submucosa of PP
Dome
John Clancy Jr., PhD
29
• Partially encapsulated and covered by epithelium.
Present in lamina propria and submucosa of
certain areas of GI tract.
 Peyer’s Patches (Ileum) (Fig. 1.20, 10.19
and 14-40)
- Villi and Domes which overlie lymphoid
tissue
- Domes are covered by M cells: Epithelial
cells which become antigen transporting.
They are derived from surface lining
epithelium and require B cells for their
development. Also found in the lung.
Dome
Wheater’s 6th Edition, Fig 11.17
30
Janeway 6th Edition
 Appendix at the beginning of the large
intestine.
- No villi but lamina propria and
submucosal lymphoid tissue .
31
Reactive Appendix
John Clancy Jr., PhD
Janeway 7th Edition fig. 1.7
Palatine Tonsil
Crypt
John Clancy Jr., PhD
32
Palatine Tonsil
a = cyst
c = crypt
John Clancy Jr., PhD
Palatine Tonsil: Stratified squamous non-keratinizing epithelia
John Clancy Jr., PhD
• Tonsils
 Palatine: lined by stratified squamous
nonkeratinizing epithelium. Between
palatoglossal and palatopharyngeal
folds.
- Many (10-12) deep crypts
- Capsule underneath
- Skeletal muscle deep to capsule
33
 Pharyngeal (Adenoids): pseudostratified
ciliated columnar with some stratified
squamous; no crypts but shallow pleats.
Roof of nasal pharynx.
 Lingual: stratified squamous, single crypt.
Palatine Tonsil
John Clancy Jr., PhD
• Mucosal Associated Lymphoid Tissue
(MALT) is found under all epithelial linings
of mucosa (respiratory, gut, urinary, etc.)
- Gut Associated Lymphoid Tissue
(GALT) is thus only a part of the
MALT system
34
Janeway 6th Edition
• Lymphocytes circulate from the
blood into various tissues and
lymphoid organs and back to the
blood continuously. 5 x 1011
leave the circulation to enter the
spleen and return into the blood
per day.
35
• Lymphocytes leave the blood thru HEV in
lymph nodes and Peyer’s Patches. They
eventually enter efferent lymphatics and
the thoracic duct system which joins the
left subclavian and internal jugular veins.
O.3 X 1011 return to the blood from the
lymph per day.
• Lymphocytes enter the white pulp
of the spleen from the marginal
zone blood sinuses. They
eventually find their way into the red
pulp blood sinuses and leave via
the splenic vein.
36
HOST DEFENSE
LYMPHOID TISSUE AND ORGANS
Note: Slides in brackets are optional.
Date: March 13, 2012
10:30 AM – 12:30 PM
Dense lymphoid tissue, i.e., aggregations of lymphocytes and other cells of that line, may
appear in small patches within organs or as the major component of certain so-called
lymphoid organs.
Like bone marrow, lymphoid tissue is actually a variety of connective tissue composed of
a fixed cell and fiber framework and a large population of free cells in various stages of
maturation. As with the granulocytes, monocytes and erythrocytes of bone marrow, it is
true of the lymphocytes of lymphoid tissue: their appearance in the blood or body tissues
in abnormal numbers or as immature stages is diagnostic of specific diseases such as
leukemia or lymphoma.
NOTE: As you proceed through the following study of lymphoid tissue, find answers to:
Where do T and B lymphocytes differentiate and proliferate? Where are their “homing
areas” in these organs? Where do plasma cells originate and where are they found in
different lymphoid tissues?
DIFFUSE LYMPHOID TISSUE – Slide #134 (appendix) and #137. Much of the wall
is infiltrated with dark, small lymphocytes. Occasionally these cells are arranged in
round nodules (follicles), but most of them are diffuse, with no particular arrangement.
PEYER’S PATCHES - Slide #132 (ileum) and DMS 134. There are groups of
lymphoid nodules that are characteristic of the wall of the lower small intestine,
primarily the ileum. In this particular instance they have merged more or less into one
large lymphoid mass, but if you look closely along the outer edge or the mass, you can
see indications of the rounded contours or originally discrete nodules. Where would you
find M cells?
NOTE:
What is meant by mucosal associated lymphoid tissue (MALT)?
What is meant by skin associated lymphoid tissue (SALT)?
PALATINE TONSIL – Slide #42 and DMS 125. Here you will notice a large diffuse
mass of lymphoid tissue, with scattered nodules. Furthermore, if you look closely, you
will see stretches of stratified squamous epithelium lining crypts which extend into the
substance of the tonsil from the epithelium lining the pharynx. Often this epithelium is
all but obscured by lymphocytes passing through it to the lumen. What is the function of
1
the tonsils? What do you suppose might be the basis of inflamed tonsils? Do any of the
nodules on your slide have germinal centers? If so, what does that indicate?
PHARANGEAL TONSIL – DMS 124
THYMUS – Slide #43 (infant), [DMS 117, DMS 118]. Here you see the thymus as it
exists in the late fetus and through early childhood. Notice that it characteristically has a
dense lymphoid cortex and a pale medulla, that it has no nodules, and that its substance is
incompletely divided into lobules by thin connective tissue septa. In the medulla, where
lymphocytes (thymocytes) don’t obscure the stromal framework, look for the larger, paler
epithelioid cells which are unique to the thymus. Although these epithelioid cells are
stellate in shape and thus form a supportive stromal meshwork (cytoreticulum) that
resembles reticular tissue, they are epithelial in origin instead of mesenchymal, contact
each other via desomsomes, and have secretory (endocrine) function as they secrete
cytokines necessary for thymocyte development. Note that even though you can only see
them in the medulla, they are present throughout the thymus.
In the thymic medulla are round, pink-stained Hassall’s corpuscles consisting of
concentrically arranged epithelioid cells, sometimes hyalinized in appearance. These are
diagnostic of the thymus.
SLIDE #44 and [DMS 119] shows remnants of the thymus as found in the adult. What
kind of tissue has mostly replaced it? You should be able to recognize the remnants of
lymphatic tissue, and in pieces of medulla you will still find Hassall’s corpuscles.
Adding up these clues, you should be able to identify such a section as “adult thymus”.
Even though it looks atrophied, is it still functioning?
LYMPH NODE – Slide #34 and DMS 120, [DMS 121]. (As you look at the structure
of a node, and then compare it with the structure of other lymphoid organs, try to select
those particular features which are diagnostic for purposes of identification.)
In Slide #34, stained with H&E, note under low power that the lymphoid tissue is
arranged in an outer cortex (containing many nodules) an inner paracortex (containing
mainly dense, diffuse lymphatic tissue) and a central medulla composed of cords of
densely packed lymphocytes). Lying between the denser areas of lymphocytes runs a
whole interconnected system of wide, paler-looking sinuses, through which lymph
“percolates” (or is filtered) as it passes through the node. Identify subscapsular
(marginal), trabecular (cortical) and medullary sinuses.
Lymph nodes are set in the path of lymph vessels; thus you may see afferent lymphatics
entering the peripheral capsule of the node, as well as efferent lymphatics (possibly filled
with lymphocytes) leaving the hilus of the node. Lymph entering via the afferent vessels
must pass through the system of sinuses before leaving via efferent vessels.
2
The primary entry ports for recirculating lymphocytes are the high endothelial
postcapillary venules (HEV) found in the deep (inner) cortex. Look for some examples.
Where do the lymphocytes go after homing to and traversing the HEV into the substance
of the node? READ about the circulation of cells through the lymph node; also about the
“homing areas” for B-cells, helper T-cells, other T-cells, plasma cells. What happens
histologically during an immune response?
Look at the cortical lymphoid nodules and notice that sometimes the cells are more
densely packed at the periphery of the sphere than in the center. Such a pale center is
called the germinal center and it is the site of active proliferation of B lymphocytes.
Can you identify a dark zone of centroblasts and a light zone of centrocytes? Look at the
comparative density of small versus large lymphocytes (centrocytes and centroblasts) in
different parts of a nodule. Look also in the medullary region of the node, where
lymphocytes are less dense, and try to find examples of small and large lymphocytes,
plasma cells and reticular cells.
SPLEEN – Slides #39 [#40, #41, DMS 122], DMS 122A, [DMS 123]. Unlike the
preceding lymphoid organs, the spleen has a large share of circulating blood components
as well as lymphoid tissue. The other organs are vacularized and, as such, have blood
vessels passing through them like any other organ, but nowhere else do the red blood
cells leave the blood stream and mingle with lymphoid elements as they do in the spleen.
Thus, you will see “white pulp” (dense lymphoid tissue) scattered throughout “red
pulp” (blood sinusoids mixed with diffuse lymphoid tissue) and other hemopoietic
elements. There is a collagenous connective tissue capsule surrounding the spleen, and
projections of this as trabeculae into the substance of the organ provide the major
framework. As with lymph node, the delicate supporting framework for all the free cells
is a combination of stellate reticular cells and fine reticular fibers. The sinusoids here, as
in bone marrow, are lined with endothelial cells which have a discontinuous basal lamina
underneath them, through which blood cells can easily pass. Macrophages in the red pulp
remove worn out red blood cells; indeed, the spleen is often called the “graveyard of red
blood cells”.
Consult your textbook for a diagram of the vascular pattern within a splenic lobule and
the arrangement of the lymphoid white pulp in relation to these blood vessels.
Understand these relationships, even though the sequences of branchings, do not clearly
in a histological section.
Look at your slides under low power to get a sense of what features identify this organ.
Notice particularly the capsule (along at least one or two edges), the scattered thick
trabeculae (pink) and the scattered lymphoid “nodules” (white pulp, stained dark blue or
purple). Although the white pulp often takes a nodular form, it must be remembered that
actually the white pulp forms periarterial lymphoid sheaths (PALS) which accompany
the central arteries along much of their length and, therefore, can sometimes be cut
longitudinally as well as transversely. Notice that most sections of white pulp contain a
central arteriole, so called, although it is actually usually eccentrically placed. This
arteriole typically has a very narrow lumen and a fairly thick connective tissue and
3
muscle sheath which stains pink in your slides. The presence of this arteriole within
white pulp is diagnostic in the identification of spleen. Look for the marginal blood
sinus and outer marginal zone which separates white from red pulp.
If the venous sinusoids of the red pulp are distended with blood (stained either pale or
bright pink), they will help in identification; look for them particularly under the capsule
or near the trabeculae. (Spleen is a brittle tissue to cut, so you may find many fine cracks
in your section).
Slide #39 is particularly good for seeing macrophages. Look for cells containing bright
yellow hemosiderin, the remnants of broken-down hemoglobin of phagocytized red blood
cells. Look particularly in a phagocytic (marginal) zone separating white from red pulp;
look also in the red pulp cords of Bilroth.
4
Katherine L. Knight, Ph.D.
Host Defense 2012
Antigen Recognition by B Lymphocytes: Immunoglobulin Structure
ANTIGEN RECOGNITION BY B LYMPHOCYTES:
IMMUNOGLOBULIN STRUCTURE
Date:
Thursday, March 15, 2012
Friday, March 16, 2012
LEARNING GOAL
You will be able to compare and contrast the structure, effector functions and antigenic features of the
five classes of immunoglobulins.
OBJECTIVES
To attain the goal for these lectures you will be able to:
●
List the classes and subclasses of immunoglobulins (Ig).
●
Diagram the basic structure of Ig (including polymeric Ig) including H and L
chains, domains, CDR, antigen binding site, Fab and Fc.
●
Describe the major biological characteristics of each Ig class/subclass.
●
Describe the major structural differences between IgG subclasses.
●
Define monoclonal antibody and describe the principle for developing them.
●
Define idiotypes and describe how antibodies can be used to treat disease.

State the half-life of serum antibody titer after disease or vaccination
READING ASSIGNMENT
Janeway et. al., 2008, Chapter 3, pp. 1110-122; 160-166
Chapter 9, pp. 400-403
Appendix p748-749
LECTURER
Katherine L. Knight, Ph.D.
Page 1 of 14
Katherine L. Knight, Ph. D.
Host Defense 2012
Immunoglobulin Structure
CONTENT SUMMARY
I. Classes of Immunoglobulins: IgG, IgA, IgM, IgD, IgE
II. Basic Immunoglobulin Structure
Core Structure
Heavy chains
Subclasses
Light chains
Domain Structure - variable and constant regions
Complementarity Determining Regions (CDR)
Three Dimensional structure
Fab and Fc fragments
III. Major characteristics of immunoglobulin classes (antibody binding and effector functions)
IgM, IgG, IgD, IgE, IgA
IV. Half-life of serum antibody titers following disease or immunization
V. Clinical uses of immunoglobulins
Polyclonal vs. monoclonal antibodies (mAb)
Treatment of B lymphoma
 Anti-CD20 mAb
 Anti-idiotype
Definition
Treatment
Treatment of other tumors with mAb
2
Katherine L. Knight, Ph. D.
Host Defense 2012
Immunoglobulin Structure
IMMUNOGLOBULIN STRUCTURE
I.
CLASSES OF IMMUNOGLOBULINS
●
●
●
●
●
II.
IgG - predominant Ab induced in secondary response
IgA - predominant Ig in external secretions
IgM - predominant Ab induced in primary response
IgD - found mainly on surface of B cells
IgE - involvement in allergic hypersensitivities
BASIC IMMUNOGLOBULIN STRUCTURE
●
CORE STRUCTURE
All antibodies have a common core structure of two identical light chains (about 24 kilodaltons [kD]) and
two identical heavy chains (about 55 or 70 kD) (Fig. 1). One light chain is attached to each heavy chain,
and the two heavy chains are attached to each other. Both the light chains and the heavy chains contain a
series of repeating, homologous units, each about 110 amino acid residues in length, which fold
independently in a common globular motif, called an immunoglobulin domain.
3
Katherine L. Knight, Ph. D.
Host Defense 2012
Immunoglobulin Structure
HEAVY CHAINS
The H chains determine the Ig isotype, IgA, IgG, IgM, IgD, or IgE.
Ig Class
Heavy Chain Type
Ig Subclass
Heavy Chain Type
IgM
mu (µ)
IgG1
(µ) 1
IgG
gamma ()
IgG2
() 2
epsilon ()
IgG3
() 3
alpha ()
IgG4
( ) 4
delta ( )
IgA1
() 1
IgA2
( ) 2
IgE
IgA
IgD
LIGHT CHAINS
All antibody light chains fall into one of two classes or isotypes,  and . Each member of a light-chain
isotype shares complete amino acid sequence identity of the carboxy terminal C region with all other
members of that isotype. In man, antibodies with  and. light chains are present in about equal number.
DOMAIN STRUCTURE
Studies of myeloma proteins resulted in identification of variable (V) and constant (C) Ig domains.
L chain has 2 domains: 1 VL domain and 1CL domain.
C-terminal half = Constant Domain
(C = constant domain of a  chain)
N-terminal half = Variable Domain
(V = variable domain of a  chain)
H chain has either 4 or 5 domains: 1 VH domain and 3 or 4
CH domains; H chain may also contain a hinge region.
4
Katherine L. Knight, Ph. D.
Host Defense 2012
Immunoglobulin Structure


Variable Region: The combination of the variable region of the H chain and the
variable region of the L chain make up the antigen binding site of an immunoglobulin.

Constant Region: Other effector functions of Ig are carried out by the constant domains.
These include the ability to cross the placenta, sites for attachment to Fc receptors of
macrophages, monocytes, mast cells and sites for binding complement.
COMPLEMENTARITY - DETERMINING REGIONS (CDR) AND THREE
DIMENSIONAL STRUCTURE OF Ig
Most of the amino acid sequence variation among different heavy and light chains that as part of
an Ig molecule bind different antigens, is confined to three separate locations in the V region.
These are called hypervariable segments, or CDRs. When VH and VL regions fold into an Ig
domain, the CDRs are present on the surface as projecting loops (Fig. 3.5); VH and VL CDRs are
in close proximity in 3D. Sequence differences contribute to variation in the chemical surface of
the loops.
The amino acid sequences of the CDRs are primarily responsible for the specificity of antigen binding.
BASIC 3D Ig FOLD
CDRs
Figure 3.5 Immunobiology 7 (Garland Science 2007)
Antibody binding site:
Figure 3.8 Immunobiology 7 (Garland Science 2007)
5
Katherine L. Knight, Ph. D.
Host Defense 2012
Immunoglobulin Structure
Fab and Fc FRAGMENTS
Fab
Fc
III.
MAJOR CHARACTERISTICS OF THE FIVE IMMUNOGLOBULIN CLASSES
/’;……………..
6
Katherine L. Knight, Ph. D.
Host Defense 2012
Immunoglobulin Structure
7
Katherine L. Knight, Ph. D.
Host Defense 2012
Immunoglobulin Structure

IgM
-
Ab of 1o response for protein Ag administered parenterally.
Major class of Ab elicited by T-independent antigen (polysaccharide).
H chains have 4 CH domains, 1 VH domain; are CHO rich.
Pentameric in its secreted form, m.w. = ~ 900,000 daltons.
J chain (mw = 15,000 daltons) joins the subunits
Expressed on the surface of B-cells as a monomer
Effective complement fixation - classical pathway.
Does not cross the placenta of man or other species.
Serum concentration = ~ 100 mg/dl in adult.
Part of B cell receptor (BCR)

IgG
-
Ab class characteristic of 2o response for most protein (T-dependent) Ag.
H chains have 3 CH domains, 1 VH domain.
Monomeric in its secreted or membrane form; m.w. = ~150,000 daltons.
Variability in complement fixation according to subclass.
Crosses the human placenta due to the presence of FcRn on the placenta.
Serum concentration = ~1200 mg/dl in adult.

IgD
-
Usual form is the membrane form; very few IgD plasma cells.
Functions as B-cell Ag receptor along with IgM
Serum concentration of adults = ~3 mg/dl
IgE
-
Class of antibody involved in allergic hypersensitivities, such as ragweed allergy.
IgE Ab(s) fix to mast cells and basophils via an Fc receptor on the cells
Has 4 CH domains and 1 VH domain.
Secreted as a monomer; m.w. ~190,000 daltons; CHO rich.
Very low concentrations in serum of normal adults (~30 µg/dl).


IgA
-
Predominant Ig class in external secretions; occurs as a dimer.
J chain joins the two subunits
Predominant form in serum is monomeric; serum IgA is ~160,000 daltons.
Function of serum IgA is not clear.
H chains have 3 CH domains, 1 VH domain.
Does not fix complement by classical pathway; can usually fix complement by
alternative pathway.
Does not cross placenta.
Serum concentration = ~300 mg/dl in adult.
Key role in mucosal immunity
8
Katherine L. Knight, Ph. D.
Host Defense 2012
Immunoglobulin Structure
Goldsby et al (2003) Immunology, WH Freeman 5th edition
Immunoglobulin levels in human serum.
The neonate is protected by passively transferred maternal IgG for the first few months after
birth. Adult levels of IgM are reached at about 10 months of age; IgG at 4 years; IgA at about 10
years of age.
IV.
Half-life of serum antibody titers after immunization or disease.
9
Katherine L. Knight, Ph. D.
Host Defense 2012
Immunoglobulin Structure
Serum spanning a 20 year peiord was analyzed to determine the hal-life of antibody to
infectious agents following immunization or infection.
Result: The half-life of serum antibody titers ranged from 11 yrs for tetanus to 3014
years for measles.
Taken from: Amanna et
al. (2007) N Engl J Med
357:1903
V. CLINICAL USES OF IMMUNOGLOBULINS
EPITOPE: antigenic determinant; many associated with each protein antigen molecule
USING ANTIBODIES TO DISTINGUISH Ig CLASSES
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Katherine L. Knight, Ph. D.
Host Defense 2012
Immunoglobulin Structure
POLYCLONAL VS MONOCLONAL ANTIBODIES
Polyclonal antibody is found in the serum of immunized individuals. Each of the different
antibodies present in the serum, directed against different epitopes, are made by separate
clones of B lymphocytes (plasma cells). Single B cells that make a single kind of
antibody (monoclonal) can be immortalized in vitro and used as a continual source of a
specific monoclonal antibody.
Anti- heavy chain (anti-IgM) can be used to identify B lymphocytes

TREATMENT OF B LYMPHOMA WITH ANTIBODIES
o

Anti-CD20, a molecule uniquely present on B cells, can be used to treat patients with B
lymphoma.
Idiotypes - The term idiotype was used to describe the "unique" set of antigenic
determinants found in and about the antigen combining site of an antibody. The CDRs
can themselves become antigenic determinants (idiotopes). Idiotypes are detected by
anti-idiotype antibodies.
B
B
B
B
B
11
B Lymphoma: Expansion
of a single clone of B cells
with same VH and VL
regions (i.e., same idiotype)
Treatment with anti-idiotype
antibody would affect only this
clone of B cells.
Katherine L. Knight, Ph. D.
Host Defense 2012
Immunoglobulin Structure
Tumor-specific Monoclonal Antibodies
Tumors treated with monoclonal antibodies
STUDY QUESTIONS
1.
List Ig classes and subclasses and their respective heavy chains.
2.
Define domain, V domain, C domain, and hinge region.
12
Katherine L. Knight, Ph. D.
Host Defense 2012
Immunoglobulin Structure
3.
Draw the domain structure of heavy chains, kappa light chains and lambda light chains.
4.
Draw a schematic diagram of IgG, IgD, IgE, monomeric IgM, polymeric IgM, monomeric IgA
and secretory IgA.
5.
Compare the similarity of heavy and light chain domains of antibodies of different specificities
(for example, anti-salmonella versus anti-pneumococcus antibodies).
6.
Identify the regions of IgG that comprise Fab and Fc fragments.
7.
Identify major effector function(s) of each immunoglobulin isotype.
8.
Compare the relative levels of each Ig class in serum and in secretions.
9.
State the relationship of VH to VL in the 3D structure of Ig.
10.
Define epitope as it relates to protein antigens.
11.
Describe how monoclonal antibodies are made and how they can be used to treat disease.
12.
Define idiotype and explain how anti-idiotype antibody can be used to treat B-cell lymphoma.
ADDITIONAL QUESTIONS
1.
is the predominant serum immunoglobulin.
2.
is the predominant secretory immunoglobulin.
3.
Most B lymphocytes have membrane Ig of what class (es)?
4.
There is approximately
5.
Antibody fragment with a single combining site is designated ________
6.
The Fc portion of the  -heavy chain has how many domains?
times more IgM than IgE in serum of normal individuals.
ANSWERS TO ADDITIONAL QUESTIONS
1.
IgG ; 2. IgA ; 3. IgD, IgM ; 4. 1,000 ; 5. Fab ; 6. CH2, CH3.
EXAMPLE OF TEST QUESTION
Ig classes can be distinguished on the basis of:
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Katherine L. Knight, Ph. D.
Host Defense 2012
Immunoglobulin Structure
A.
Their H chains.
B.
Their L chains.
C.
Both their H and L.
D.
J chain.
E.
V Regions.
CORRECT ANSWER TO ABOVE QUESTION: A
14
BASIC Ig
STRUCTURE
Immunoglobulin Isotypes
(Classes and Subclasses)
1
HYPERVARIABLE REGIONS (CDR)
3D STRUCTURE OF
Ig V AND C DOMAINS
ANTIBODY BINDING SITE (CDRs)
2
SHAPES OF
ANTIBODY BINDING SITES
Fab
Fc
Ig CLASSES (ISOTYPES)
Fab
Fc
3
POLYMERIC STRUCTURE OF IgM
STRUCTURE OF SECRETORY IgA
Secretory
Component
IMMUNOGLOBULIN CLASSES
4
IMMUNOGLOBULIN FUNCTIONS
Cross-linking of IgE
on mast cell leads to
degranulation
SERUM Ig LEVELS
DURING DEVELOPMENT
5
Half-life of Serum Antibody Titers
Amanna et al (2007) NEJM 357:1903
EPITOPES
H-chains Distinguish Ig Isotypes
Anti-μ, Anti-γ, Anti-α, etc
6
Clinical Uses of Antibodies
EPITOPE
= ANTIGEN-BINDING
POLYCLONAL
vs
MONOCLONALSITE
ANTIBODIES
• Protein Antigens have many epitopes
• B cells make Ab to a single epitope
• Different clones of B cells make Ab
to different epitopes
Polyclonal and Monoclonal
Antibodies
7
Treatment of B lymphoma with Antibodies
B Lymphoma:
Expansion of a
single clone
(blue) of B
cells with same
VH and VL
regions (i.e.,
same idiotype) B
B
B
B
B
B
B
B
Treatment with anti-idiotype
antibody would affect only
this (blue) clone of B cells.
Tumor-specific Monoclonal Antibodies
Tumor antigens and
monoclonal antibody treatment
8
IVIG
Intravenous Immunoglobulin
Used to treat many
inflammatory diseases
Clinical Uses of IVIG
Nimmerjahn and Ravetch Ann Rev Immunol (2008) 26:513
Mechanism of action of IVIG
Nimmerjahn and Ravetch Ann Rev Immunol (2008) 26:513
9
Fc
Activating and Inhibiting Fc Receptors
on macrophages
Nimmerjahn and Ravetch Ann Rev Immunol (2008) 26:513
•The sialic acid-rich fraction of IVIG has
enhanced anti-inflammatory activity
• >30 glycovariants found in human serum
• IgG antibodies from human arthritis
patients have decreased level of
terminal sialic acid residues
Nimmerjahn and Ravetch Ann Rev Immunol (2008) 26:513
10
Nimmerjahn and Ravetch Ann Rev Immunol (2008) 26:513
How to Generate Millions of Ig?
VH
VL
VH
VL
Germline Organization of Ig Genes
11
Ig Constant Region Genes
Ig Gene Rearrangements
Ig Gene Rearrangements
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Antigen-Receptor Genes and B Cell Development
ANTIGEN-RECEPTOR GENES
& B CELL DEVELOPMENT
Date: Friday, March 16, 2012
10:30 AM – 11:30 AM
Monday, March 19, 2012
10:30 AM – 11:30 AM
LEARNING GOAL
You will be able to diagram and describe the stages of B cell development including somatic DNA
rearrangements, and explain how a large antibody repertoire is developed. You will also be able to
diagram the structure and rearrangement of T cell receptor (TCR) genes and explain how a large TCR
repertoire develops.
OBJECTIVES
To attain the goal for these lectures you will be able to:
 State the approximate size of antibody repertoire needed for immune protection.
 Diagram the order of H and L chain gene rearrangements that occur during B cell
development.
 Describe the mechanism by which individual B cells make only one antibody (allelic
exclusion)
 Diagram the sequence of events leading from rearrangements of germline Ig genes to
production of an Ig molecule.
 Describe how antibody diversity is generated through combinatorial joining, junctional
diversity, and N segment addition.
 Describe how antibody diversity is generated in secondary lymphoid tissues
 Describe the maturation of B cells from stem cells, including the pre-B cell receptor.
 State the cell origin of CLL, Burkitt’s lymphoma, Hodgkin’s lymphoma, ALL, and
multiple myeloma
 Draw the structure of αβ and γδ T cell receptors.
 Diagram the mechanism by which T cells express a single antigen receptor
READING ASSIGNMENT
Janeway’s Immunobiology (2008), Chapter 4; Chapter 7, pp 262-272; 308-312
LECTURER
Katherine L. Knight, Ph.D.
1
Katherine L. Knight, Ph.D.
Host Defense 2012
Antigen-Receptor Genes and B Cell Development
CONTENT SUMMARY
Introduction
Genetic basis for the antibody repertoire
I. Germline organization of Ig genes
Three groups of genes; κ,λ, and H chain
V, D and J gene segments
Cκ and CH region genes
II. Ig gene rearrangements - basic features
B-cell development
Order of rearrangements
Heavy chain: DJ, VDJ
Light chain: VJ
ProB, PreB, Immature B and Mature B cells
Allelic exclusion - only one VH and one VL rearranged/B cell
Mechanism of Ig gene rearrangement
Conserved heptamer/nonamer (RSS) recognition sequences
Membrane IgM vs. secreted IgM
Surface IgM and IgD
IIII. Generation of Antibody Diversity in bone marrow
Combinatorial joining of V, D and J gene segments
Junctional diversity- N-region addition and imprecise joining
Combinations of H and L chain proteins
Selection against self-reactive B cells
IV. Generation of antibody diversity in secondary lymphoid tissues
Somatic mutation
V. B-lineage tumors: Leukemia and lymphoma
Chromosomal translocations
VI. T cell antigen receptor
Structure
Gene organization and rearrangement
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Antigen-Receptor Genes and B Cell Development
INTRODUCTION
Problem: One can estimate that an individual needs to be able to make antibodies to recognize about one
million epitopes. What is the genetic basis for being able to generate so may different antibodies?
Major rule of antibody synthesis: A single B cell makes only one kind of antibody specificity (one VH
and one VL), i.e., allelic exclusion occurs. Also, a single plasma cell makes only one kind of antibody;
i.e., 1 kind of H chain & 1 kind of L chain; B cells may violate this rule & synthesize two or more heavy
chain isotypes simultaneously for the cell surface, eg. IgM and IgD.
I.
GERMLINE ORGANIZATION OF IMMUNOGLOBULIN GENES
Three groups of Genes: κ, λ and H Chain
The genes for the immunoglobulin polypeptide chains (and for the T-cell receptor chains)
are split, Ig genes undergo a process of somatic DNA recombination (rearrangement)
during B cell ontogeny.
Each of the 3 gene families, the kappa light chain family, the lambda light chain family
and the heavy chain family can be divided into V-region genes and C-region genes. The
κ, λ and H chains are located on separate chromosomes. Each set of genes, κ, λ and
heavy chain, has a similar basic organization.
A. V, D and J Gene Segments
The Ig heavy and light chain loci are composed of multiple genes that give rise to the V
and C regions of the proteins, separated by stretches of non-coding DNA. At the 5'
end of each Ig locus are the V region exons, each about 300 base-pairs (bp) long,
separated from one another by non-coding DNA of varying lengths. Downstream of the
V genes are additional coding sequences, 30 to 50 bp long, which make up the joining
(J) segments and, in the H chain locus only, the diversity (D) segments. The J and D
gene segments code for the carboxy terminal ends of the V regions, including the third
hypervariable (complementarily-determining) regions of antibody molecules. Thus, in
an Ig light chain protein (κ or λ), the variable region is encoded by the V and J exons and
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Antigen-Receptor Genes and B Cell Development
the constant region by a C exon. In the heavy chain protein, the variable region is
encoded by the V, D, and J exons. The constant region of the protein is derived from the
multiple C exons and, for membrane-associated heavy chains, the exons encoding the
transmembrane and cytoplasmic domains.
B. C Region Genes
At varying distances 3' of the V genes are the C region genes. In both mouse and man,
the κ light chain locus and a single Cκ gene and the genes for heavy chain C regions (CH)
of different isotypes are arranged in a tandem array. Each heavy chain C region gene
actually consists of three to four exons (each similar in size to a V region exon) that make
up the complete C region, and smaller exons that code for the carboxy terminal
transmembrane (TM) and cytoplasmic domains of the heavy chains.
II.
IMMUNOGLOBULIN GENE REARRANGEMENTS - BASIC FEATURE
All cells except B-lineage, including plasma cells contain Ig genes in
the germline configuration. The Ig genes are expressed only in B-lineage cells.
Rearrangements of Ig genes are the essential first steps in the production of antibodies.
A.
B Cell Development
Order of Ig gene rearrangement and B cell development
DNA rearrangements occur in a precise order and occur independent of antigen
stimulation.
1.
Heavy chain - DJ. The first Ig gene rearrangement involves the heavy chain
locus and leads to joining of one D and one J gene segment with deletion of the
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Katherine L. Knight, Ph.D.
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Antigen-Receptor Genes and B Cell Development
intervening DNA.
2.
Heavy chain - VDJ. Following the DJ rearrangement, one of the many V genes
is joined to the DJ complex, giving rise to a rearranged VDJ gene. At this stage,
all D segments 5' of the rearranged D are also deleted. This VDJ recombination
occurs only in cells committed to become B lymphocytes and is a critical control
point in Ig expression because only the rearranged V gene is subsequently
transcribed. The C region genes remain separated from this VDJ complex by an
intron.
3.
Light chain - VJ. The next somatic DNA recombination involves a light chain
locus. One V segment is joined to one J segment, forming a VJ complex, which
remains separated from the C region by an intron, and this gives rise to the
primary RNA transcript. Splicing of the intron from the primary transcript joins
the C gene to the VJ complex, forming an mRNA that is translated to produce the
κ protein. The light chain assembles with the previously synthesized μ to form
the complete membrane IgM molecule, which is expressed on the cell surface,
and the cell is now the immature B lymphocyte.
ProB Cells: These cells are precursors of PreB cells. They have IgH DJ gene rearrangements
and no light chain gene rearrangements.
Pre B Cells: All B lymphocytes arise in the bone marrow from a stem cell that does not
produce Ig. The earliest cell type that synthesizes a detectable Ig gene product contains
cytoplasmic -heavy chains composed of variable (V) and constant (C) regions. This
cell is called the pre-B lymphocyte and is found only in hematopoietic tissues, such as
the bone marrow and fetal liver. The pre-B receptor is comprised of surrogate light
chain, -chain, Ig and Ig
Immature B Cells: At the next identifiable stage in B cell maturation,  or λ light chains
are also produced. These associate with μ heavy chains and then the assembled IgM
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Antigen-Receptor Genes and B Cell Development
molecules are expressed on the cell surface, where they function as specific receptors for
antigens. IgM-bearing B cells that are recently derived from bone marrow precursors are
called immature B lymphocytes because they do not proliferate and differentiate in
response to antigens. Once a B cell expresses a complete heavy or light chain, it cannot
produce another heavy or light chain containing a different V region.
Mature B Cells: Having acquired a complete Ig and, therefore, an antigen specificity, B
cells migrate out of the bone marrow and can be found in the peripheral circulation and
lymphoid tissues. They continue to mature, even in the absence of antigenic simulation.
Mature B cells co-express μ and δ heavy chains in association with the original κ or λ
light chain and, therefore, produce both membrane IgM and IgD. Both classes of
membrane Ig have the same V region and hence the same antigen specificity. Such cells
are responsive to antigens.
B. Allelic Exclusion: Only one IgH and one IgL allele are productively rearranged.
Each B cell clone and its progeny are specific for only one antigenic determinant. It is,
therefore, necessary for each B cell to express only one set of Ig heavy and light chain V
genes throughout its life. This occurs because only one functional heavy chain VDJ and
one functional VJ gene rearrangement occur in each cell. The expression of only one
allele in a cell is termed allelic exclusion.
C. Mechanism of Ig gene rearrangement

Conserved recognition sequences
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Antigen-Receptor Genes and B Cell Development
The recombination of V, D and J gene segments is mediated by specific DNA
recognition sequences (RSS) located in the intervening DNA 3' of each V exon and 5' of
each J segment and flanking both sides of each D segment . The RSS are highly
conserved stretches of seven or nine nucleotides separated by non-conserved 12 or 23
nucleotide spacers. In a light chain gene, each heptamer or nonamer adjacent to a V exon
recognizes a complementary stretch adjacent to a J exon. This allows recombinase to
bring the two exons together, forming a loop of intervening DNA.
Enzymes then excise the intervening DNA in this loop and anneal the ends of the V and J
exons. Recombination is mediated by recombinase enzymes RAG1 and RAG2. Not all
rearrangements are functional. The junctions are in CDR3.
Membrane IgM vs. Secreted IgM : Secreted and membrane forms of -chain result from alternative
RNA splicing
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Antigen-Receptor Genes and B Cell Development
Co-expression of membrane IgM and membrane IgD on B cells: IgM and IgD on a given B
cell have the same idiotype (VH + VL). Given that one cell makes only one antibody, how can μ and
δ heavy chains be produced simultaneously by the same B cell? The answer is,  and  chains with the
same VH domain result from alternative splicing of primary transcripts (nuclear RNA).
III.
GENERATION OF ANTIBODY DIVERSITY IN BONE MARROW
A.
Combinatorial Joining of V, D and J Gene Segments
The germline contains multiple germline VH and VL genes that have different sequences
and produce Ig molecules with different specificities. D and J gene segments also
contribute to diversity.
The somatic recombination of Ig DNA participates in the generation of antibody diversity
in several ways. The combinatorial associations of different V, D, and J gene segments
lead to a large potential for generating different antibody specificities. The maximum
possible number of combinations is the product of the number of V, D (if present), and J
gene segments at each locus. Every clone of B cells and its progeny express a unique
combination of V, D, and J genes.
Even the same set of germline V, D, and J gene segments can generate different amino
acid sequences at the junctions. This junctional diversity can arise from imprecise
joining or N-region addition.
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Antigen-Receptor Genes and B Cell Development
B.
Imprecise joining: or imprecise DNA rearrangement occurs because nucleotide
sequences at the 3' end of a light chain V gene segment and the 5' end of a J gene segment
or at the ends of V, D, and J gene segments in a heavy chain can each recombine at any
of several nucleotides in the germline sequence.
C.
N-region addition: Nucleotides, called N sequences, which are not present in the
germline, can be added to the junctions of rearranged VDJ genes during rearrangement.
This addition of new nucleotides is a random process mediated by an enzyme called
terminal deoxyribonucleotide transferase (TdT).
Because of junctional diversity (due to imprecise joining and N-region addition), the number of
different amino acid sequences present in CDR3 of antibody molecules is much greater than the
number of germline V and D segments present in the genome (Table 1).
D.
Combinations of H and L Chain Proteins
In addition to these mechanisms operative at the level of Ig genes, the combination of
different H and L chain proteins also contributes to diversity because the V region of
each chain participates in antigen recognition.
Table 1. Mechanisms Contributing to the Generation of Antibody diversity in the Human
Germline genes
V gene segments
J segments
D segments
Combinatorial joining
V x J (X D)
H-L chain associations
Hx
H


40
6
25
40
5
0
30
4
0
6,000
200
120
1.2 x 106
What happens to B lineage cells in the bone marrow that have non-functional V(D)J gene
rearrangements or that express self-reactive antibody?
B cells with non-functional V(D)J genes are deleted. B cells with anti-self reactivity can
become anergic, can be deleted, or they can be rescued by receptor editing.
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Antigen-Receptor Genes and B Cell Development
IV.
GENERATION OF ANTIBOD DIVERSITY IN SECONDARY LYMPHOID
TISSUES
A.
Somatic hypermutation: Changes in antigen-binding specificity as a result of
somatic mutations in V genes can generate additional diversity.
When does somatic hypermutation (diversification) occur?
Pre B and early B cells utilize unmutated germline V genes.
Primary antibody response utilizes mostly unmutated V genes.
Secondary antibody response utilizes mostly V genes that have undergone
somatic mutation.
B. The enzyme, AID, is required for somatic hypermutation. AID deficient patients have only
IgM and the Ig genes are not somatically diversified.
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Antigen-Receptor Genes and B Cell Development
V.
B-LINEAGE TUMORS
Individual B-lineage cells in BM or periphery can undergo neoplastic transformation giving rise
to leukemia or lymphoma.
Chromosomal translocation
of c-myc into the IgH locus
leads to B cell tumor.
B-lineage tumors
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Antigen-Receptor Genes and B Cell Development
VI.
T CELL ANTIGEN RECEPTORS (TCR)
Most T cells have αβ TCR; ~5% of T cells have γδ TCR
A.
STRUCTURE
The αβ TCR is a disulfide linked heteradenic of α and β chains
(α = 45 kD;  = 40 kD).
-
Each chain has 2 Ig - like domains; The N-Terminal domains are variable (V)
regions; The C-Terminal domains are polypeptide constant regions
The overall structure of δγ TCR is similar to αβ except that the polypeptide
chains are designated γ and δ.
B. GENE ORGANIZATION AND REARRANGEMENT
-
The overall organization of TCR genes is similar to that of Ig genes
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Antigen-Receptor Genes and B Cell Development
-
The V regions of α and γ chains are encoded by V and J gene segments.
The V, D and J gene segments are associated with the same conserved heptamer
and monomer nucleotide sequences found in Ig genes. Thus, TCR genes
rearrange by the same mechanism as Ig genes.
Most importantly - as in B cells, only one VDJ and one VJ gene rearrangement
occur in each T cell. Therefore, each T cell expresses a single TCR, specific for
one particular antigenic determinant.
Comparison of Gene Rearrangements in B and T Cells
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Antigen-Receptor Genes and B Cell Development
STUDY QUESTIONS
1.
Identify the approximate number of heavy chain V, D and J gene segments in the germline.
2.
Identify the approximate number of light chain V and J gene segments in the germline.
3.
Diagram the organization of germline V, D and J heavy chain gene segments and V and J light
chain gene segments. (Be sure to identify the exons and introns within each diagram).
4.
State the number of CH genes and their relationship to Ig subclasses.
5.
List the order of Ig H and L chain gene rearrangements.
6.
Diagram Ig H and L chain gene rearrangements.
7.
What is the significance of allelic exclusion and describe the mechanism responsible for
generating allelic exclusion.
8.
Given a B cell with a VDJ gene rearrangement describe the fate of:
1.
VH gene segments upstream of the VDJ gene.
2.
D gene segments that were not used in the VDJ gene rearrangement.
3.
J gene segments downstream of the VDJ gene.
9.
Describe how the hepatamer/nonamer signal sequences (RSS) mediate V, D and J gene
rearrangements.
10.
State the role of RNA processing in synthesis of Ig L chains.
11.
For the light chain genes, compare the intervening nucleic acid sequences that are lost during
DNA recombination with those lost during RNA splicing.
12.
Diagram the sequence of events in B cells that lead to the production of L chains; start with
rearrangement of L chain genes.
13.
State how μ and δ H chain can arise from the same primary transcript.
14.
Compare the organization of TCR and Ig genes.
15.
Compare the mechanism of rearrangements of TCR genes and Ig genes.
16.
State the approximate number of antibody specificities needed for protection.
17.
State the approximate number of germline VH, VL, D, JH, and JL, gene segments.
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Antigen-Receptor Genes and B Cell Development
18.
Explain the mechanisms by which CDR3 becomes more variable than CDR1 and CDR2.
23.
T
Compare the contribution of somatic mutation to the diversity of Ig receptors with the diversity of
cell receptors.
ADDITIONAL QUESTIONS
Indicate whether each of the following statements is true or false:
1.
Immunoglobulins are encoded by genes located on one chromosome.
2.
Within one immunoglobulin molecule there may be two types of light chain.
3.
The immunoglobulin combining site (for antigen) is contributed by the hypervariable regions
within each of the heavy and light chain variable regions.
4.
IgG and IgM molecules are distinguished by differences in their heavy chain constant region
sequences.
5.
Myeloma proteins are the result of polyclonal B cell activation.
6.
V gene segments are not joined with J gene segments in cells other than lymphoid cells.
7.
Allelic exclusion refers to the phenomenon where only the heavy or light chain is produced by
the cell but not both.
8.
In pre-B cells, both heavy and light chain genes are rearranged.
ANSWERS TO THE ADDITIONAL QUESTIONS
1.
False. Immunoglobulins are encoded by 3 gene families: for heavy chain, kappa and
lambda light chains. The 3 gene families each reside on a separate chromosome.
2.
False. The light chains of a single antibody are identical and the heavy chains are
identical also. Therefore, one immunoglobulin may be either lambda or kappa.
3.
True.
4.
True.
5.
False. Myeloma proteins are homogenous and result from 1 B cell becoming concerous.
6.
True.
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Antigen-Receptor Genes and B Cell Development
7.
False. Allelic exclusion means that only one allele of each gene (H, and kappa or
lambda) is expressed at the protein level - i.e., either the maternal or paternal
heavy chain allele is expressed - as well as that for either kappa or lambda. The
result of allelic exclusion is the expression by the B cell of only one
immunoglobulin and one immunoglobulin specificity (clonal expression).
8.
False. Light chain genes are in germline configuration.
EXAMPLES OF TEST QUESTIONS
1.
Somatic hypermutation is most active during:
A.
Differentiation of pre-b cells into mature B cells
B.
Differentiation of pre-T cells into mature T cells
C.
Generation of memory B cells
D.
VH, D, JH gene rearrangements
E.
A primary immune response
Match the following Ig gene rearrangements with the appropriate cell type:
A.
DJ.gene rearrangement on one allele; VDJ gene rearrangement on the other allele;
no VJ gene rearrangement
B.
VJ gene rearrangements on both kappa-chain alleles.
C.
VDJH gene rearrangement on one allele; VJL gene rearrangement on one allele.
D.
No VDJ, DJ or VJ Ig gene rearrangements.
1. Plasma cell.
2.
Pre-B lymphocyte.
3.
T-lymphocyte.
Answers to above questions: 1-C
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Antigen-Receptor Genes and B Cell Development
2-A
3-D
17
How to Generate Millions of Ig?
VH
VH
VL
VL
Germline Organization of Ig Genes
Ig Constant Region Genes
1
Ig Gene Rearrangements
Ig Gene Rearrangements
B Cell Development
in Bone Marrow
HSC
HSC =
hematopoietic
stem cell
(CD34+)
2
B cell Development
in Bone Marrow
HSC
B cell Development
in Bone Marrow
Allelic
Exclusion
3
How Does Ig Gene
Rearrangement
Occur?
Conserved Recombination Recognition
Sequences for RAG-1 and RAG-2
Mechanism
of Ig Gene
Rearrangement
Loop-out
Excision
Re-joining
4
How to Obtain
Secreted IgM and
Membrane IgM
with the Same VDJ region?
&
How to Obtain IgM and IgD
with the
Same VDJ region?
Ans: Alternative RNA Splicing
5
Co-expression of
Membrane IgM and IgD
IgM
IgD
Generation of Antibody Diversity
How large a repertoire do we need?
How, genetically, is such a large
antibody repertoire generated?
Generation of the Antibody Repertoire
1. Combinatorial V(D)J gene joining
2. Junctional diversity- (imprecise
joining)
3. N nucleotide addition by TdT
4. Combinatorial association of
H and L chains
6
Junctional Diversity During Vκ and
Jκ Joining (Imprecise Joining)
N-Region Addition
N-Region
Addition
N-Region Addition
(N-nucleotides)
VH
DH
JH
TdT (terminal deoxyribonucleotide transferase)
DH
VH
AGCGT
N
JH
TACGCAG
N
7
Mechanisms Contributing to Ab Diversity
Germline Genes
V gene segments
J segments
D segments
λ
H
κ
40
40
6
5
4
25
0
0
6,000
200
120
30
Combinatorial
Joining
VxDxJ
H-L Chain Associations
1.2 x 106
Hxκ
What if V(D)J gene
rearrangements
are non-functional?
Ans: They die
or encode
self-reactivity?
Elimination of Self-reactive B cells
8
A Second Chance for
Self-reactive
B cells:
Receptor
Editing
Receptor Editing of Anti-self
VJ gene rearrangements
9
Immature B Cells Leave
Bone Marrow to Periphery
Periphery
Antigen
Immune Response to
the Antigen
Somatic Hypermutation of Ig Genes
Affinity Maturation
Required Enzyme =
AID
B-lineage Tumors
10
B cell tumors
developing from
chromosomal
translocation of
c-myc into the IgH
locus
Structural
Similarity
of TCR and Ig
Organization of TCR Genes
11
Rearrangement of TCR Genes
T cell
Rearrangement
Excision Circles
(TRECS)
Gene Rearrangements: T cells vs B cells
12
Germinal Centers – Sites of Somatic
Hypermutation
Germinal Centers Induced During
an Immune Response
Germinal Center Reactions
1. Somatic hypermutation
a. AID is required
2. Development of memory B cells
3. Development of plasma cells
13
B Cell Development
in Bone Marrow
14